Nitric Oxide Synthase Assay Kit | NOS3 assay kit

Ultra Sensitive Assay for Nitric Oxide Synthase

Cat. Num. AAA480447

• Gene Names: NOS3; eNOS; ECNOS
• Synonyms: Nitric Oxide Synthase; Ultra Sensitive Assay for Nitric Oxide Synthase; Ultrasensitive Colorimetric NOS Assay; NOS3 assay kit

• Sequence Length: 118

• Preparation and Storage: 1. Store this kit and its components according to the label.; - The Nitrate Reductase, NADPH Part A and NADPH Part B should be stored at At -20 degree C.; - - The reconstituted Nitrate Reductase may be stored at At -20 degree C for up to six months.; - - The diluted standard curve may be stored at 4 degree C for up to 24 hours.; - - All other kit components should be stored at 4 degree C.

Related Product Information for NOS3 assay kit

Principle of the Assay: NADPH and L-arginine are required for the continual operation of NOS and production of nitric oxide (NO). In aqueous solution, NO rapidly degrades to nitrate and nitrite. Spectrophotometric quantization of nitrite using Griess Reagent is straightforward, but does not measure nitrate. This kit employs recombinant nitrate reductase (NaR) for conversion of nitrate to nitrite prior to quantization of nitrite using Griess reagent — thus providing for accurate determination of total NOS activity. This kit can be used to accurately measure as little as 1 pmol/uL (~1uM) NO produced in aqueous solutions. Very little sample is required (5 to 100 uL depending on the [NO] in the sample). The completed reaction is read at 540 nm.

Background/Introduction: The traditional method for measuring nitric oxide synthase (NOS) activity is performed by radiochemical assay that measures the conversion of L-[3H]arginine to L-[3H]citrulline. This method is expensive and requires regulation of radioactive materials. The Ultrasensitive Colorimetric NOS Assay Kit is a low-cost novel assay that allows for the detection of NOS activity without the need for radioactivity. Our Ultrasensitive NOS Assay Kit employs a NADPH recycling system to allow NOS to operate linearly for hours as nitric oxide-derived nitrate and nitrite accumulate. NOS can be assayed spectrophotometrically by measuring the accumulation of its stable degradation products, nitrate and nitrite. The ratio of these two products in biological fluids, tissue culture media, etc. may vary substantially. Hence, for accurate assessment of the total nitric oxide generated, one must monitor both nitrate and nitrite. An excellent solution to this problem is the enzymatic conversion of nitrate to nitrite by the enzyme nitrate reductase (NaR), followed by quantization of nitrite using Griess Reagent. This kit allows for efficient highthroughput screening of NOS activity in resting cells or cell lysates as well as biological fluids and tissue homogenates. The kit is also ideal for in vitro NOS assays using recombinant purified NOS. All materials necessary to perform the entire assay in a 96-well microplate format are provided with the kit.

References

1. Ghigo, D., et al. (2006). Nitric Oxide 15:148-153
2. Schmidt, H.H., et al. (1995). Biochemica 2:22-23

NCBI and Uniprot Product Information

• NCBI GI #: 4262036
• NCBI GeneID: 4846
• UniProt Accession #: P29474; Q13662; Q14251; Q14434; Q548C1; Q6GSL5; Q9UDC6; A0S0A7; A0S0A8; A8KA63; B2RCQ1; E9PFR2
• Molecular Weight: 67,876 Da
• NCBI Official Full Name: nitric oxide synthase, partial
• NCBI Official Synonym Full Names: nitric oxide synthase 3 (endothelial cell)
• NCBI Official Symbol: NOS3
• NCBI Official Synonym Symbols: eNOS; ECNOS
• NCBI Protein Information: nitric oxide synthase, endothelial; EC-NOS; NOS type III; NOSIII; cNOS; constitutive NOS; endothelial NOS; nitric oxide synthase 3 transcript variant eNOS-delta20; nitric oxide synthase 3 transcript variant eNOS-delta20-21; nitric oxide synthase 3 transcript variant eNOS-delta21
• UniProt Protein Name: Nitric oxide synthase, endothelial
• Protein Family: Nitric oxide synthase
• UniProt Gene Name: NOS3
• UniProt Synonym Gene Names: cNOS; eNOS; NOSIII
• UniProt Entry Name: NOS3_HUMAN

NCBI Description

Nitric oxide is a reactive free radical which acts as a biologic mediator in several processes, including neurotransmission and antimicrobial and antitumoral activities. Nitric oxide is synthesized from L-arginine by nitric oxide synthases. Variations in this gene are associated with susceptibility to coronary spasm. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2009]

Uniprot Description

eNOS: endothelial constitutive nitric oxide synthase. Synthesizes nitric oxide (NO) from arginine and oxygen, which is implicated in vascular smooth muscle relaxation through a cGMP-mediated signal transduction pathway. NO mediates vascular endothelial growth factor (VEGF)-induced angiogenesis in coronary vessels and promotes blood clotting through the activation of platelets. Induced in humans by caloric restriction. Stimulated by calcium/calmodulin. Inhibited by NOSIP and NOSTRIN.

Protein type: Oxidoreductase; Amino Acid Metabolism - arginine and proline; EC 1.14.13.39

Chromosomal Location of Human Ortholog: 7q36

Cellular Component: Golgi membrane; cytoskeleton; cytoplasm; plasma membrane; caveola; nucleus; cytosol

Molecular Function: calmodulin binding; actin monomer binding; protein binding; FAD binding; nitric-oxide synthase activity; NADPH-hemoprotein reductase activity; FMN binding; iron ion binding; heme binding; NADP binding; cadmium ion binding

Biological Process: regulation of systemic arterial blood pressure by endothelin; negative regulation of potassium ion transport; negative regulation of muscle hyperplasia; positive regulation of vasodilation; nitric oxide metabolic process; negative regulation of cell proliferation; regulation of the force of heart contraction by chemical signal; negative regulation of hydrolase activity; regulation of blood pressure; lipopolysaccharide-mediated signaling pathway; negative regulation of blood pressure; angiogenesis; regulation of blood vessel size; nitric oxide biosynthetic process; interaction with host; mitochondrion organization and biogenesis; negative regulation of calcium ion transport; in utero embryonic development; positive regulation of guanylate cyclase activity; regulation of sodium ion transport; ovulation from ovarian follicle; positive regulation of angiogenesis; response to heat; arginine catabolic process; endothelial cell migration; blood vessel remodeling; regulation of nitric-oxide synthase activity; blood coagulation; vascular endothelial growth factor receptor signaling pathway; nitric oxide mediated signal transduction; lung development; smooth muscle hyperplasia

Disease: Preeclampsia/eclampsia 1; Alzheimer Disease; Hypertension, Essential; Stroke, Ischemic